P1183: CAR-T MORPHOLOGICAL CHARACTERISTICS AND DYNAMICS IN PERIPHERAL BLOOD, EVALUATED BY A NOVEL MICROSCOPY PLATFORM
Authors: G. Fridberg, B.-Z. Katz, D. Benisty, C. Perry, R. Ram, I. Avivi
Journal: HemaSphere (2022)

Background: Chimeric Antigen Receptor T-cells (CAR-T) therapy becomes a preferable therapeutic approach in patients with relapsed and refractory (R/R) non-Hodgkin’s large B cell lymphomas (LBCL) who failed ≥2 therapeutic regimens. However, there are no commercial assays for routine CAR-T measurements and clinical studies often employ PCR or FACS, both expensive and relatively complicated. Detection of CAR-T in peripheral blood smear (PBS) is challenging, mostly due to the lack of data regarding their morphology prior to transfusion and the low sensitivity of currently available technologies. As oppose to the currently available digital morphology, Scopio Labs Full Field Morphology (FFM) is a novel digital microscopy platform that provides high-resolution images combined with a full field analysis, incorporating artificial intelligence capabilities, thereby enabling the detection and classification of rare cells in PBS, including CAR-T cells. Aims: The dynamics of CAR-T quantity and subtypes in PBS were evaluated, and correlated with responsiveness to treatment and treatment-related toxicities. Methods: Morphological library of CAR-T cells was established from cells obtained directly from the CAR-T transfusion-bag (Fig. 1A). FFM analysis of consecutive PBS samples obtained from 26 R/R DLBCL patients, treated either with tisagenlecleucel (tisa-cel) or axicabtagene ciloleucel (axi-cel) at the Tel Aviv Sourasky Medical Center between October 2019-October 2020 was performed. Results: We identified in the CAR-T cell transfusion bags 5 distinct CAR-T morphological sub groups prior to transfusion: regular, activated, apoptotic, multinucleated and cells in mitosis (Fig. 1A). First, 79 smears, obtained from 18 patients, were examined with the FFM platform vs the CellaVision DM1200 system. Overall, 24,247 WBC were detected by the FFM platform (average 303 cells per PBS), compared with 4,335 detected by CellaVision DM1200 system (average 54.4 cells per PBS). Detection of CAR-T was superior with the FFM platform (median difference per PBS - 2, IQR 0-6, p<0.001). Then, PBS from 26 patients, 21 (80.7%) treated with tisa-cel, and 5 (19.2%) treated with axi-cel, were analyzed by the FFM platform. The absolute number of CAR-T cells in the PBS post infusion was higher in patients treated with axi-cel (across days 1 to 15). The average numbers of total, regular, and activated CAR-T cells, measured on day 5 post CAR-T-transfusion, were all higher in patients that obtained CR vs those who did not (Fig. 1B). Longer duration of cytokine release storm (CRS) was associated with higher number of CAR-T cells with activated morphology, and a lower presence of apoptotic cells on day 14. Higher number of apoptotic cells on day 14 were associated with a deferred CRS onset, while lower number of activated morphology cells on day 14 was associated with earlier onset of CRS. There was a positive correlation between the number of cells, measured on day 7 by FACS, and time to peak of regular morphological CAR-T cells (r=0.67, p=0.034, n=10). Image:Summary/Conclusion: In this study, we show that CAR-T cells has heterogeneous morphology identified in PBS. Specific morphological features in specific days were associated with outcome and therapy related toxicities, emphasizing that CAR-T monitoring require quantitative, qualitative and repeated measurements. Thus, morphological CAR-T surveillance using FFM might serve as an available, simple and inexpensive method to provide clinically relevant insights.